Histones are important in the organization of DNA into a chromatin structure and in the retrieval of genetic information. Specific modifications on histones regulate gene activity, leading to either expression or silence (1,2). Of the modifications in the euchromatins of eukaryotes that have been examined, Histone 3-Lysine 4 (H3-K4) trimethylation is recognized as a hallmark of transcriptionally active genes (3). It is believed that trimethylated H3-K4 is a recognition site for the recruitment of additional factors required for transcription (4,5). Abnormalities in H3-K4 methylating enzymes have been observed in various cancers (6,7), the most prominent example of which is Mixed Lineage Leukemia (MLL) (8), which is also known as MLL1, ALL-1, HRX, and HTRX1.
MLL is enzymatically active in a multiprotein complex and acts as both a global and a specific gene regulator (9,10). The most well-known targets for MLL are the homeobox (Hox) genes such as Hox-a9 and Hox-c8. These genes encode for a class of homeodomain transcriptional factors that regulate organ formation during embryo development, as well as proper hematopoiesis in adults (11-13). Increased expression levels of certain Hox genes, accompanied by MLL aberrations, such as gene fusion and amplification, are frequently observed in acute leukemias, such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) (14-16). Injection of cells overexpressing Hox-a7 and Hox-c8 into nude mice results in well vascularized tumors in 4-5 weeks (17). Abnormal Hox gene expression also is observed in solid tumors, such as prostate carcinoma and primary colorectal tumors (18,19). MLL therefore is a promising therapeutic target for several forms of leukemias and solid tumors.
Immediately after translation, MLL is proteolytically cleaved to yield 180-kDa C-terminus (MLL1C) and 320-kDa N-terminus fragments (MLL1N) (20). These are assembled together in a multi-subunit complex together with several other proteins, including WD Repeat Domain 5 (WDR5), Absent Small or Homeotic-Like (Ash2L), and Retinoblastoma Binding Protein 5 (RbBP5), each of which is a common component of all known human H3-K4 methylating complexes.
MLL forms a catalytically active core complex with WDR5, RbBP5, and Ash2L that can dimethylate H3-K4 in vitro (21). Although MLL alone can minimally partially monomethylate H3-K4, all the other members of the core complex are required for dimethylation, including WDR5, which forms a bridge between MLL and the remainder of the core complex. In the absence of WDR5, MLL is unable to associate with RbBP5 and Ash2L, and fails to dimethylate H3-K4 in vitro (21,22). Knock-down of WDR5 is known to result in a significant decrease in the levels of H3-K4 trimethylation and expression of Hox-a9 and Hox-c8 genes in 293 cells (23). Blocking of the WDR5-MLL interaction therefore is an effective strategy for inhibiting MLL activity.
It recently has been shown that MLL binds to WDR5 via an arginine (Arg) (residue 3765) containing sequence (24,25), which is similar to that used by the N-terminal of H3 in its interaction with WDR5(26-29). WDR5 has a canonical conformation that contains a central cavity, and both H3 and MLL peptides use an Arg residue to interact with this cavity through the arginine binding site. Although crystal structures show that H3 and MLL peptides have very similar binding modes to WDR5 in this arginine binding site, MLL peptides have a higher affinity to WDR5 than H3 peptides (30). The MLL-derived, 12-residue WIN (WDR5 Interacting Motif) peptide (residues 3762-3773) (Table 1) has been shown to dissociate MLL from the remainder of the complex in vitro (21). The WIN peptide therefore represents a starting point for the design of inhibitors to block the interaction of MLL with WDR5.
TABLE 1Sequence of WIN peptide and N-terminus of H3 peptide. Residues 1-10in H3 and 3762-3773 in MLL1 are shown. The numbering assignedbelow compares the residues in these two peptides.WINGSARAEVHLRKSN-term of H3ARTKQTARKSNumbering−2−112345678910used hereinAbbreviations:G—Gly—Glycine;S—Ser—Serine;A—Ala—Alanine;R—Arg—Arginine;T—Thr—Threonine;E—Glu—Glutamic acid;K—Lys—Lysine;V—Val—Valine;G—Gln—Glutamine;H—His—Histidine;L—Leu—Leucine.